Method and device for the selective withdrawal of components from complex mixtures

ABSTRACT

The method according to the invention permits a selected withdrawal of one or a few molecularly disperse or cellular components of a system, such as molecules, molecular complexes, vesicles, micelles, cells, optionally together with an associated volume element V having a size of 10 −9  l≧V≧10 −18  l from a larger sample volume. The selected transfer of the sought component to another environment is effected by defining the space and time of withdrawal by means of a signal correlating with the small component to be withdrawn. The method is particularly useful for the withdrawal of non-abundant components the existence of which can be detected in a preceding step by a scanning process. The method is also useful for the withdrawal of per se unidentified components.

[0001] The present invention pertains to a method for the withdrawal ofone or a few components of a system, a device for performing saidmethod, and uses thereof.

[0002] The functional characterization of single molecules or molecularcomplexes, viruses or individual cells is possible by means of themethods described in Rigler et al. (PCT/EP 94/00117). By means of themethod described, information can be obtained about whether, in acomplex mixture of a solution or suspension or in a two-dimensionallayer, there are contained within very small volume elements (<10⁻¹² l)individual molecules or molecular complexes which are subject toparticular interactions with defined target molecules.

[0003] For many analytical and synthetic problems, it is already a greatadvantage to know that a sought molecule does exist in a mixture beinganalyzed. Frequently, however, the problem to be solved by the presentinvention arises, that is to selectively remove the. molecule oncerecognized as being the desired one in terms of a preparativeenrichment, e.g., in order to avoid or to facilitate cloning steps.Another problem in particularly diluted solutions having a concentrationof 10⁻¹² M is to quickly find a volume element as a part of the samplevolume in which a representant of the sought substance is present. It isby no means necessary that the sought substance be known. When unknownpathogens or active substances are sought, it may be that onlyinteractions with known substances are known, or interactions withoptionally present detector molecules can be postulated.

[0004] Methods and devices have been described for the withdrawal ofwhole cells. The corresponding devices are known as cell sorters. Forexample, certain cell types of a blood picture can be identified fromparticular parameters of light scattering or fluorescence, and the cellsof a defined specification counted. The cells can be stained withfluorescence-labeled antibodies and differentiated by their surfaceantigens or classsified by hybridization methods (in situ) due to theirnucleic acid contents. After their isolation in droplets, the cells areanalyzed in flow and may be selectively separated or fractioned byselective electrostatic deflection of single droplets. Appropriatedevices are commercially available and are of great importance inclinical diagnostics and research. PCT/EP 93/03077 discloses a methodfor separating individual, linearly separated volume elements from acapillary flow.

[0005] The object of the present invention is to provide a method whichpermits to withdraw a small volume element of a solution or suspensionfrom a larger volume element of a sample volume of said solution orsuspension at defined space coordinates wherein said small volumeelement contains a target substance. Such a small volume element can bedefined using analytical methods as described in the Rigler et al.application (PCT/EP 94/00117). This involves analyzing extremely smallmeasuring volumes as a part of a larger, surrounding sample volume byconfocal illumination of a volume element, excitation by the light usedfor said illumination, and/or registration of specific fluorescencesignals, and concluding therefrom the presence of particular components.Alternatively, said illumination may also be achieved, for example, bythe method of near field spectroscopy using apertures which are smallerthan the wavelength of the irradiated electromagnetic radiation. Theobject, to selectively withdraw a sought, detected substance, aparticular molecule, a molecular complex or a cell due to their spectralproperties, is also to be achieved.

[0006] The method according to the invention may be employed toparticular advantage for detecting as yet non-identified, unknownpathogens or immunogens. (Unknown) pathogens or immunogens can becharacterized and optionally obtained in a preparative manner. Aparticularly advantageous procedure according to the invention makes useof the fact that pathogens will first proliferate after having infecteda host organism without being prevented therefrom by a ready immunedefense. Only after a certain period of time has elapsed, the immunedefense will establish a humoral immune response, e.g., by the synthesisof various immune globulins (principally IgM, followed by IgG), andlater a cellular immune response. Patients suspected to have undergonean infection or contact with an as yet unknown pathogen or immunogenwithout detectability of known antigen properties with respect to aparticular reaction or detectability with known antiserums/antibodiesare the starting material for pathogen isolation. Later, in the phase ofchronic disease, it is considered that antibodies against the suspectedpathogen, e.g., a virus, will have formed in the meantime with the viruslevel, however, having largely decreased. Serums from this phase of thedisease serve to prepare the immuneglobulins.

[0007] The fraction of immuneglobulins normally contains only a minorpercentage of antibodies directed against the unknown pathogen. Themajor part of the antibodies is directed against a variety of antigenstructures which are not related with the sought specificpathogen/immunogen. Therefore, it is more difficult to characterize thepathogen through an immune complex using serums from one single patient.

[0008] However, the sought immune complex can be isolated by recurringto some expected characteristics:

[0009] The immune complex contains the mass-determining major portion ofa pathogen the mobility of which is between that of a small RNA virusand that of a bacterium.

[0010] The pathogen has several antigenic binding sites which areoccupied by more than one dye-labeled antibody.

[0011] In a preferred procedure (FIG. 5), the antibodies from twopatients in the condition of the hypothetically chronic phase can beprepared separately and labeled with different dyes or with dyes to bedifferentiated by cross correlations. Said at least two patients areselected such that there is a high probability that both patients havebeen infected by the same pathogen. Since pathogens usually carry alarge number of antigenic determinants on the surface/viral envelope, itis likely that the immune complexes formed after reaction with a mixtureof differently labeled antiserums will simultaneously comprise thedifferent labeling dyes.

[0012] The experiments are performed as illustrated in FIG. 5.

[0013] Detection of Individual Bacteria by the Binding Specifities ofSurface-Expressing Bacteria

[0014] For a large number of important applications in modernbiotechnological research, it would be extremely advantageous andefficient if the detection of a functional biomolecule in a given samplevolume in a method could be replaced by the detection of an individualbacterium or virus having functional surface proteins. The decisiveadvantage is the interesting coupling of a phenotypical expressionproduct, e.g., a natural or recombinant surface protein, to its geneticconstruction plan. The method, in particular, is to be seen in thecontext of a preparative use according to the invention by means ofwhich cells or molecular complexes determined to be the sought ones canbe separated from an environment.

[0015] Determination and Preparative Recovery of the ImmunogenicEpitopes of Microorganisms

[0016] The genome of microorganisms comprises about 10⁷ nucleotides.Subgenic fragments having an average length of 100 amino acids can beexpressed according to the method described by shot-gun expression.Taking into account the variation of reading frames (factor 3) and anassumed non-coding counter-strand, 10⁸ recombinant bacterial clonescontain 100 copies of each segment. 10⁸ recombinant bacterial clones arecontained in 1 ml of a suspension of 1 OD which can be examinedindividually within about 24 hours, by the method according to theinvention, e.g., in terms of their binding properties with IgE or IgEbearing cells from an allergic patient. According to the invention, thecorrespondingly characterized bacteria are separated, or at least highlyenriched, biologically expanded, or the corresponding genome segment isamplified and characterized by enzymatic amplification methods.

[0017] If a corresponding bacterium is detected in the measuring volumeelement, it can be immediately withdrawn from the mixture, according tothe invention, by sucking the volume element surrounding thebacterium/molecular complex through a capillary or separating themolecular complex by electrophoresis, electroosmosis or dipole induction(FIGS. 1 and 2).

[0018] An idea on which the invention is based is to employ anelectrically, optically or mechanically controlled sucking device theaperture of which is large as compared to the measuring volume, butsmall as compared to the dimensions of the sample volume, in order toachieve the object of the invention. According to the invention, theelectrical, optical or mechanical pumping system is controlled by anFCS-controlled pulse-generator so that a small fraction of the samplevolume is separated from the total volume in such a way that a backdiffusion can be essentially excluded. This is achieved byelectrophoresis, induced dipole moments, electroosmosis, mechanicallyinduced pressure jump, or the pressure of light. The method permits thewithdrawal of one or a few components of a system, such as molecules,molecular complexes, vesicles, micelles, cells, optionally embedded inan associated volume element (withdrawal volume), V (10−9 l≧V≧10⁻¹⁸ l).This volume element is part of a larger volume of an environment whichcontains the small components to be withdrawn (sample volume). Thewithdrawal is effected by transferring the component or components toanother environment wherein space and time of the withdrawal aredetermined by an analytical signal which is correlated with the smallcomponent to be withdrawn. The analytical methods which may be used arethose by which the molecular contents of smallest volume elements (10⁻¹⁴l ) can be analyzed as described in the International Application ofRigler et al., PCT/EP 94/00117. The sample volume is connected with areceptor means through a pore of a capillary or a pore of a membranewall whose smallest aperture D is given by 100 μm≦D ≦0.1 μm.

[0019] In a preferred embodiment, a capillary is employed as describedin Neher et al., Methods in Enzymology, vol. 207, 3-14. This capillaryis connected, e.g., with a conveying means, such as a pump, which iscapable of drawing off the solution volume surrounding the molecularcomplex. It is preferred according to the invention to use amechanically, light pressure or electrically controlled sucking systemor a (piezo/isolenoid) pump controlled dispenser system.

[0020] Several withdrawals to the same receptor means can be performedin one or more steps wherein the individual withdrawal processes may beperformed independently in terms of a gathering process.

[0021] In many cases, the electrically controlled withdrawal through adirected transport of the volume element by means of at least oneelectrical voltage or field strength impulse is preferred. Otherembodiments operate mechanically by means of at least one pressuredifference impulse and/or by means of at least one light pressureimpulse in the direction of the pore aperture using methods aspublished, e.g., by Weber and Greulich, Int. Rev. Cytol., 1992, 133,pages 1-41. The receptor means may be a capillary the lumen of which ispreferably larger than the diameter of the pore or capillary tip theaperture of which is in direct contact with the sample volume. Inconstantly thin capillaries, electroosmosis can be performed as iscommon in capillay elektrophoresis since otherwise, in mechanical volumetransport, the flow resistance could become disadvantageously large.

[0022] In the case of an electrically mediated withdrawal, the soughtcomponent in the measuring volume is selectively transferred to thereceptor means preferably by means of an electrical.. field strengthimpulse by shortly applying an electrical field at least once forelectrophoresis of electrically charged components and/or forelectroosmosis with coupled transport of electrically neutral molecules.One electrode may be placed in electrically conducting contact with thesolution on the side of the sample volume while the other electrode isplaced in electrically conducting contact with the solution on the sideof the receptor means and the conducting contact between the twocompartments is established through the pore. When the withdrawal iseffected by means of a well-aimed pressure pulse, the desired transportis caused by at least one short increase of the pressure in the volumeoutside the receptor compartment as compared to the pressure inside thereceptor compartment, and/or by a short reduction of the pressure insidethe receptor compartment. Per se known dispensers (Microdrop) orpumping/sucking devices (solenoid pumps, stepping motor controlledpumps) have been found to be useful. Volume elements as small as ≦1 nlcan thereby be transferred from a larger volume of a solution orsuspension to a receptor compartment by transferring the small volumeelement through a pore having a diameter of ≦100 μm wherein the time andthe space coordinate of the withdrawal process is controlled by acorrelated analytical system, such as fluorescence correlationspectroscopy (FCS).

[0023] Especially a reduced pressure pulse from a piezo-controlleddispenser module the filling volume of which is inside the receptorcompartment, and/or a pressure pulse and/or reduced pressure pulsecauses that the volume of the receptor means is enlarged, or the samplevolume is diminished in favor of the receptor means. According to theinvention, transport to the receptor volume ensues. The size of thereceived volume is controlled by the number of the dispensed droplets orthe steps of the stepping motor or the length and intensity of the lightpressure pulse.

[0024] The hardware/software-coupled on-line operating analyzing systemtriggers the withdrawal time through the received correlating signal.The withdrawal is performed in the moment when the particle or particlesto be withdrawn is/are present within the withdrawal volume with highprobability. This needs not necessarily occur in a virtuallysimultaneous manner with the reception of the positive analyticalresult. When a molecule, molecular complex, virus or cell has beenidentified by FCS as a component to be withdrawn, it may be withdrawnimmediately or later. This is based on the assumption that therespective component

[0025] either is present, due to forced transport as described orelectrophoretical migration, in a particular place at a defined timeshortly after the measurement and can be separated from thereelectrophoretically or mechanically according to FIGS. 1 and 2; or,

[0026] when there is no forced translation, is still present near thedetection volume from which the component can be separatedelectrophoretically, optically by light pressure, or mechanically.

[0027] Commercial cell sorters are equipped with a system for theisolation of cells following prior analysis which may be coupled,according to the invention, with the analytical method of fluorescencecorrelation spectroscopy. Usually, cells are guided in a liquid-coatedcapillary flow through a cuvette which is coupled to a conventionalfluorescence measuring device and/or a light-scattering measuringdevice. In a defined space and time interval behind the measuringdevice, a standardized continuous division of the thin liquid jet intoindividual exiting droplets is provided at the end of the capillary byapplying a continuous sound frequency. When a cell of a desired celltype passes the cuvette, this cell will be present in one of thesedroplets after a defined period of time which droplet can be selectivelydeflected from its trajectory after the isolation by means of a coupledsignal, e.g., by means of a pulse in an electrostatic field, and thuscan be separated in a separate receptor means. The drawback of thismethod resides in the fact that in a flowing stream, only an integralfluorescence signal in terms of a pure intensity measurement can beobtained. In contrast, the coupling with the method of fluorescencecorrelation spectroscopy in small volume elements according to theinvention enables a differentiation of the fluorescence signals obtainedby their assignment to different molecular sizes and mobilities. This isrequired, e.g., for distinguishing receptor-bound ligands from freeligands present. According to the invention, the method of fluorescencecorrelation spectroscopical detection of fluorescing particles, such asmolecules, vesicles, cells or molecular complexes, in a mechanicallyinduced capillary flow is employed with a selective. sorter device forwithdrawal through a pore.

[0028] The actual separating process may take place after the passage ofindividual volume elements through the pore by transferring themeasuring volumes yielding a positive registration with an associatedvolume element to separate receptor means following a defined space andtime correlation.

[0029] In another preferred embodiment, the FCS measuring process mayalso take place prior to the exit from the pore of a capillary tip whichis coupled with a microdispenser means wherein small measuring volumeswith an associated volume element of the environment are collected asdroplets in different receptor means by various mechanical orfield-induced deflections.

[0030] In particular, the time and/or space specific correlation betweenthe analysis of a subvolume of the sample volume (measuring volume)within volume element V and the withdrawal of a desired component byremoving the volume element V is effected with the aid ofhardware/software. It is effected in such a way that at least onecomponent which has been positively identified in volume element V willbe present in the withdrawn volume element V during the withdrawalprocess. The pore of the receptor means is mechanically approached tothe volume element, and/or the volume element V or parts thereof aretransported to the pore of the receptor compartment with a predeterminedtime correlation by transport in the flow or by electrostatic ormagnetic field gradients. The analysis may also be performed immediatelyin front of the pore of the receptor compartment. Preferably, thesubvolume (measuring volume) is smaller than the volume element V. Thisincreases the probability that the positively identified component iswithdrawn before it has moved too far out of the measuring volume.

[0031] The analytical method must meet certain requirements in orderthat it may be employed in the present method. For example, the signalcorrelating with the withdrawal process is detected by an opticalanalytical system which is capable of analyzing specific molecularproperties in volume elements as small as <10⁻¹⁴ l. This is preferablydone by analysis systems based on confocal laser correlationspectroscopy or fluorescence correlation analytics based on near fieldspectroscopy the signal of which time-controls a selective withdrawalprocess on-line and in a software-controlled fashion. A number ofcoupled analytical and withdrawal processes can be sequentially employedin a cascade-like manner according to the invention wherein thewithdrawn sample volumes are subsequently again subjected to an analysiswith or without an intermediate dilution step, and are again withdrawnafter completion of the analysis in enriched form by a second and/orfurther withdrawal unit.

[0032] Such components can be withdrawn which form a complex with atleast one reaction partner capable of being spectroscopically detected(indirectly), or which have themselves sufficient fluorescenceproperties (directly).

[0033] Of particular interest is the method in combination with theprocedure for withdrawing as yet unidentified (unknown) pathogensaccording to the invention. The object is to proliferate an isolatedpathogen in vivo or to proliferate the genetic material of the pathogenor parts thereof in vitro by amplification of the nucleic acid containedtherein in terms of a shot-gun method, and its characterization bysequencing.

[0034] Also important is the selected withdrawal of components whichhave been unknown to date with respect to their molecular nature, suchas molecules, cells, vesicles, molecular complexes, which can befunctionally identified, e.g., through an enzymatic activity or complexformation.

[0035] As outlined in FIG. 5, unknown particles, such as pathogens orimmunogens, are withdrawn according to the invention by collectingserums from at least one organism which is infected with theunidentified pathogen. At least one serum (serum 1) is obtained from thephase of an acute infection by the as yet unknown pathogen or immunogen,and at least one other serum (serum 2) is obtained from the same or atleast one other organism with the same or a homologous infection in thephase of chronical disease. The unknown pathogen or immunogen from serum1 is complexed with indirectly or immediately fluorescence-dye labeledantibodies from serum 2, and the complex formed is measured. Importantin this connection is cross-correlation, such as described in PCT/EP94/00117, by which, e.g., the simultaneous binding of differentfluorescing ligands, such as antibodies from different organisms, can bemeasured. The labeling of antibodies may be done immediately by at leastone reaction with dyes capable of coupling, or indirectly by reactionwith dye-labeled antibody binding domains, in particular protein Aderivatives or protein G derivatives. The unidentified pathogeniccomponents may prove to be per se known microorganisms. The detectedcharacteristics are specific interactions with surface-expressed orcytosolic-expressed structural elements of natural or recombinantproteins or peptides or enzymatic activities with fluorescence-labeledtarget molecules.

[0036] The method according to the invention may cause the detection ofmolecules in very low concentrations. For example, scanning of fetalcells in the maternal blood may be performed. With the method accordingto the invention, very low concentrations (<10⁻¹² M) of fluorescingmolecules can be determined. However, the method may becomeimpracticable if, for establishing one or more measuring volumes, toolong waiting with unchanged space coordinates is necessary until asought molecule passes the space element of the measuring volume. Thisproblem also arises with higher concentrations (>10⁻¹² M) if thediffusion times are very short as is the case, e.g., with cells andcell-bound molecules. In such cases, the method according to theinvention may be modified so that the actual measuring process ispreceded by a scanning process in which the space coordinates arevariied in a measuring technique continuous or discontinuous in timeuntil a signal of the desired quality is detected. This may be, forinstance, the common occurrence of a correlated fluorescence with twodifferent emissions when the cross-correlation method is used. When asignal is detected, the FCS measuring process is started. The durationof a scanning process may be less than one millisecond per measuringprocess. This is sufficient to establish that the scanned measuringvolume or the measuring volumes measured in parallel do not contain thesought component. In this approach, it has to be considered that theabsolute values of the average characteristic diffusion times areinfluenced in a calculatable manner. For example, it may be that fixedmolecules (e.g. on fixed bacterial cells) directly and exclusivelyreflect the variation in time of the relative change of the spacecoordinates of the measuring volume with respect to the coordinates ofthe sample volume, or about half of the average dwelling time withmobile small molecules and abrupt changes of the space coordinates sincethe molecules are already within the measuring volume at the beginningof the measuring process.

[0037] Scanning processes preceding the actual measurement becomeimportant, e.g., when cell populations are to be analyzed wherein only afraction of the cells, molecules or molecular complexes bear theproperties which determine the withdrawal. This is the case, e.g., inthe analysis of evolutively prepared mutant populations of recombinantcells, but also in the analysis of maternal blood for the presence offetal nucleated erythrocytes which are to be analyzed for particulargenes or chromosomal anomalies.

[0038] The method is suitable for a method which will be referred to asfunctional gene extraction in the following. This means the preparationof genetic probes for the identification/detection/ cloning of specificfunctions which are encoded in a whole genome or in a cDNA library. Itsapplication may be exemplified by the functional genome analysis byusing phage or bacterial display systems, as well as correspondingapplications in evolutive bio-technology. Both examples involve thedetection and selection of cells or phages having specific bindingproperties to particular ligands before a background of non-reactingphages or bacteria.

[0039] Thus, the number of screened volume elements significantlyincreases. In combination with cross-correlation, many volume elementscan thus be screened in the μs to nanosecond range in single andmultiarray operation. The dislocation is only interrupted, e.g., whendifferently colored, correlating signals can be detected in the measuredvolume element. When this is the case, material parameters of thecomponents, e.g., translational diffusion, can be determined. This timeis statistically shorter by a time element to be calculated (about 50%)as compared to the case that a particle must penetrate into the volumeelement by itself or by forced diffusion. Once a particle is detected,it can be detected a second time by scanning the immediate environment.

[0040] According to the invention, the measuring volume may be composedof measuring subvolumes illuminated in parallel by simultaneouslyilluminating a multitude of measuring volumes by at least oneelectromagnetic radiation source using at least one holographic grid forgenerating a multitude of volume elements.

[0041] The illumination of a multitude of volume elements in parallelwith confocal optics is described in DE 40 35 799. A parallelillumination of measuring volumes the relative distances of which are inthe submicron range is not or only unsatisfactorily accomplished by thedevices described. The illuminations to be provided in the methodaccording to the invention having dimensions in the lower μm range andbelow are accomplished by using holographic grids.

[0042] Extended arrays of small volume elements can be illuminated byusing holographic grids. According to the invention, the measuringvolumes are measured confocally for fluorescence properties of moleculescontained therein by using a multitude of pinhole apertures in theobject plane, by positioning multiarray detector elements in the objectplane, or by using optical fiber bundles to which the light is coupledin the object plane and transferred to photon detectors.

[0043] In the highly parallelized illumination of small volume elements,there is the problem of registration of the emitted fluorescence signalsfrom the individual volume elements. In the patent application PCT/EP94/00117, it is reported that it is possible to illuminate small spaceelements in parallel and to focus the respective fluorescence signalsindividually on multiarray detectors by using confocal pinhole aperturesystems in the object plane, or to couple the signals into opticalwaveguides at the position and in lieu of the pinhole apertures and toguide them onto detector elements, or to position the multiarraydetectors themselves in lieu of and at the position of the pinholeapertures. There is also described the possibility to illuminate alarger volume element and to combine it with the above describedconfocal, parallel focussing of small subvolume elements.

[0044] However, in high parallelizations, the requirements on the numberof detector arrays and the computation effort associated with the datareceived in the parallel processing become considerable. According tothe invention, these problems are solved by collecting the signalsintegrated over a number of space elements with a registrating device inanother mode of coupling small space elements illuminated in parallel.This approach is especially useful in such application in which

[0045] a large number of volume elements is to be screened;

[0046] the computation effort is to be minimized in favor of thecomputing capacity employed and the computing time;

[0047] the number of the volume elements measured in one measurement andthus the total volume measured is to be maximized;

[0048] signals from molecules, molecular complexes or cells are to beanalyzed in high dilution;

[0049] the precision of the space-resolved detection is of minorimportance;

[0050] the number of the emitted light quantums during diffusion througha single space element is sufficient for a correlation.

[0051] According to the invention, at least two measuring volumes incommon or assembled in groups are focussed confocally onto at least onedetector element of a photon-registrating measuring element in theobject plane in the signal registration.

[0052] For the detection of fluorescing molecules in very lowconcentrations, the sample volume is subjected to a scanning processaccording to the invention prior to the actual measurement andwithdrawal of at least one component wherein by a continuous ordiscontinuous variation in time of the space coordinates of themeasuring volume relatively to the space coordinates of the samplevolume, the time for detecting a sought particle is shortened. The timeinterval δt for the measurement of one or more volume elements havingdefined space coordinates prior to the detection of a sought molecule byits fluorescence measuring signal is shorter than the average dwellingtime of the sought molecule within a measuring volume.

[0053] A device for performing the method is characterized in that apore of a porous receptor means is approached closely to the opticalmeasuring volume and said receptor means is connected with amechanically, optically or electrically controllable withdrawal device.It comprises the arrangement of a closed or open container for receivinga sample volume, coupled with a measuring device for the illuminationand/or measurement of a small volume element (measuring volume) byelectromagnetic radiation, and at least one connection to at least onesecond volume element in a receptor means which is in direct contactwith the sample volume through an aperture and a liquid phase whereinsaid aperture is preferably immediately adjacent to said measuringvolume.

[0054] The method according to the invention may be used, in particular,for the preparative recovery of unknown pathogens, immunogens ororganisms which functionally express parts of a genome, and for theanalysis and preparative recovery of nucleated fetal cells from maternalblood.

[0055] Such problems are connected with methods for the evolutiveoptimization of peptides and/or proteins by using mutagenesis methodsand selection methods, as suggested, for instance, in the InternationalPatent Application PCT/EP 94/00117. It is possible to screen about 10⁹bacteria for their binding properties to specific dye-labeled substanceswithin 24 hours, e.g., for the presence of a bacterium which expresses asurface protein/peptide capable of interacting with the target moleculeof a predetermined concentration. The corresponding bacterium can becloned from such a reaction mixture according to conventional methods ordirectly isolated therefrom with the method according to the invention.

[0056] Another important field of applications results from theso-called genome project for the functional mapping of gene segmentsfrom genomic libraries, cDNA libraries or libraries of subgenicstructural elements (Shape Space). In this way, genomic and/orsubgenomic segments can be determined among extended collectives bytheir functions, e.g. their functional binding behavior to targetmolecules.

[0057] The use of the described method of functional assignment ofgenetically coded peptide segments may become of great importanceparticularly in allergological research. The assignment ofimmunodominant epitopes on allergens (e.g. Aspergillus, milk protein,α-amylase) is of extraordinarily great importance and to date has been aproblem difficult to solve. Typical problems in practice are:

[0058] Determination of the IgE-binding molecules among a mixture ofsubstances which is undefined in most cases. For example, it isimportant to answer the question as to which components of soybeanlecithin are immunogenic: the pure substance alone, the pure substancein its interaction with contaminations of the preparation, or theinteraction with structures of the receptor organism. According to theinvention, the different immunogenic substances can be differentiated inthe mixture by means of labeled IgE from patients.

[0059] By the expression of subgenic gene segments,

[0060] the immunodominant epitopes can be narrowed down, characterizedand preparatively recovered with the method mentioned above. With theseresults, and with the methods described in DE 41 12 440 C2, evolutivelyanalogous functional molecules can be generated which lack thecorresponding immunodominant regions, e.g., attenuated immunogenicα-amylase or washing agent proteases;

[0061] the specific epitopes can be readily prepared according tostandard methods of genetic engineering, and employed as pure detectingreagents or used for desensibilization.

[0062] By means of the method according to the invention, certainproblems can be solved which could not be solved to date or which couldonly be solved with an unreasonably large effort.

[0063] Screening of pharmacologically active substances by the bindingof known fluorescence-labeled ligands to per se known receptors whichmay be present on cells or natural or artificial vesicle structures.

[0064] There are natural and chemically synthesized drugs withpharmacological activity whose target molecules are unknown. Thesetarget molecules can be extracellular molecules (e.g. proteaseinhibitors), surface membrane receptors (e.g. insulin), solublereceptors as mediators (steroid hormone binding receptors), or cellularsoluble structural proteins or enzymes.

[0065] Thus, according to the invention, the extremely important problemcan be solved, to find, characterize and optionally to isolate thepharmacologically important target molecule of a known drug:

[0066] search for orphan receptors;

[0067] elucidation of mechanisms of pharmacological action;

[0068] search for analogous active substances;

[0069] search and differentiation of different receptor molecules,preferably in differentiatable biological targets (different celldifferentiation, tumor/non-tumor, disease-associated,non-disease-associated, etc.).

LEGEND OF THE FIGURES

[0070]FIG. 1

[0071] (Molecule Collector)

[0072] The schematical drawing describes the principle of the device inwhich the components of a small volume element can be transferred fromcompartment A to a compartment B through a pore which is the openconnection between compartments A and B by means of a pressure orreduced pressure pulse or an electrical pulse. One part of this volumeelement is the measuring volume of <10⁻¹⁴ l represented in darkimmediately in front of the pore in which the FCS analysis takes place.A cell or a macromolecular complex may also be accessible by anappropriately directed light pressure by means of a laser pulse which isgenerated perpendicular to the pore diameter and can be directed totransport a complex recognized as a desired one to the receptor means ofcompartment B.

[0073]FIG. 2

[0074] (FCS Selection of Individual Microorganisms with SelectedFractioning)

[0075] The figure shows the FCS selection of individual microorganismsaccording to the invention with selected fractioning from a continuouslyor discontinuosuly moved sample volume. The FCS measuring volume islocated immediately in fromt of the aperture of a capillary pore and isrepresented by a hatched column within the focussing conus of the FCSillumination or near field illumination. In consecutive steps, measuringvolumes identified as positive can each be transported from the samplecontainer to the receptor means together with the surrounding volume ina computer-controlled process. This can be accomplished, e.g., byconnecting a microdrop dispenser device or, in a simple embodiment,e.g., by coupling a stepping motor controlled syringe (nlreception/step), or an electrical pulse. Thus, several volume elementscan be accumulated in the receptor means during a measurement.

[0076]FIG. 3

[0077] (Cascade Enrichment)

[0078] By serially connecting separation devices as described in FIG. 2,the separation performance can be increased. This is important, forexample, when single particles are to be separated free of background,if possible, from a highly complex mixture (10¹² particles) of, e.g.,recombinant bacteria or phages in high concentrations.

[0079]FIG. 4

[0080] (FCS Selection of Individual Microorganisms)

[0081] The figure describes a device for separating bacteria expressingparticular properties which can be measured by FCS from mixtures. Amixture of at first non-induced bacteria is fed to a capillary flowsystem. In a mixing chamber, IPTG, for example, is supplied as anexpression inducing reagent. After a sufficiently long flow time, theexpression product is supplied with an assay system comprising markermolecules which can subsequently be measured at a defined position bytheir interaction with a possible expression product. In addition, thefigure is to indicate that a positively identified measuring volume mayalso be withdrawn at a position distant with respect to space and timeprovided that the space/time coordinates are in a known and definedrelationship to one another.

[0082]FIG. 5

[0083] (Detection and Preparation of New Pathogens)

[0084] The figure demonstrates the procedure according to the inventionwhen unknown pathogens are selected which can be detected viacross-correlation by means of FCS wherein the differently labeledantibodies directed against a particular pathogen may preferably bederived from different patients.

1. A method for the withdrawal of one or a few components of a system,such as molecules, molecular complexes, vesicles, micelles and/or cells,together with an associated withdrawal volume element, V, with 10⁻⁹l≧V≧10⁻¹⁸ l, from a sample volume containing the components to bewithdrawn by transferring the component or components to anotherenvironment wherein space and time of the withdrawal are determined by asignal correlating with the component to be withdrawn which signal isestablished by an optical analytical system, in particular based onconfocal laser correlation spectroscopy or near field spectroscopy,which is capable of analyzing specific molecular properties in volumeelements as small as 10⁻¹⁴ l.
 2. The method according to claim 1,characterized in that said sample volume is connected with said otherenvironment in the form of a receptor means through a pore of acapillary or membrane wall whose smallest aperture D is defined by 100μm≧D≧0.1 μm.
 3. The method according to claims 1 and/or 2, characterizedin that at least one withdrawal to the same receptor means is performedin one or more steps wherein the individual withdrawal processes areperformed independently in terms of a gathering process.
 4. The methodaccording to at least one of claims 1 to 3, characterized in that saidwithdrawal is effected through a directed transport of said volumeelement by means of at least one electrical voltage or field strengthimpulse and/or mechanically by means of at least one pressure differenceimpulse and/or by means of at least one light pressure impulse.
 5. Themethod according to at least one of claims 2 to 4, characterized in thatsaid receptor means is a capillary the lumen of which is larger than thediameter of the pore or capillary tip the aperture of which is in directcontact with the sample volume.
 6. The method according to at least oneof claims 1 to 5, characterized in that said withdrawal is performedselectively by means of an electrical field strength impulse by shortlyapplying an electrical field at least once for electrophoresis ofelectrically charged components and/or for electroosmosis with coupledtransport of electrically neutral molecules wherein one electrode is inelectrically conducting contact with the solution on the side of thesample volume while the other electrode is in electrically conductingcontact with the solution on the side of the receptor means and theconducting contact between the two compartments is established throughthe pore.
 7. The method according to at least one of claims 1 to 6,characterized in that said withdrawal is performed selectively by meansof a mechanical pressure pulse by at least one short increase of thepressure in the volume outside the receptor compartment as compared tothe pressure inside the receptor compartment, and/or by a shortreduction of the pressure inside the receptor compartment.
 8. The methodaccording to claim 7, characterized in that the reduced, pressure pulseis caused by a piezo-controlled dispenser module the filling volume ofwhich is inside the receptor compartment, and/or the pressure pulseand/or reduced pressure pulse is caused by the volume of the receptormeans being enlarged by a change of the piston position of a coupledpiston pump device, preferably. controlled by a stepping motor, or thesample volume is diminished in favor of the receptor means.
 9. Themethod according to at least one of claims 6 to 8, characterized in thatthe size of the received volume is controlled by the number of thedispensed droplets or the steps of the stepping motor.
 10. The methodaccording to at least one of claims 1 to 9, characterized in that saidcorrelating signal triggers the withdrawal time which is in that momentwhen the particle or particles to be withdrawn is/are present within thewithdrawal volume with high probability.
 11. The method according to atleast one of claims 1 to 10, characterized in that said measuring volumeis a subvolume of said sample volume.
 12. The method according to atleast one of claims 2 to 11, characterized in that the time and/or spacespecific correlation between the analysis of said measuring volumewithin volume element V and the withdrawal of a desired component byremoving the volume element V is effected with the aid of acomputer/software means wherein at least one component which has beenanalyzed in volume element V will be present in the withdrawn volumeelement V during the withdrawal process wherein the pore of the receptormeans is mechanically approached to the volume element, and/or thevolume element V or parts thereof are transported to the pore of thereceptor compartment with a predetermined time correlation by transportin the flow or by electrostatic or magnetic field gradients, and/or theanalysis is performed immediately in front of the pore of the receptorcompartment.
 13. The method according to claim 1, characterized in thatsaid measuring volume is smaller than the volume element V.
 14. Themethod according to at least one of claims 1 to 13, characterized inthat a number of coupled analytical and withdrawal processes aresequentially employed in a cascade-like manner wherein the withdrawnsample volumes are subsequently again subjected to an analysis with orwithout an intermediate dilution step, and can be withdrawn again aftercompletion of the analysis in enriched form by a second and/or furtherwithdrawal unit.
 15. The method according to at least one of claims 1 to14, characterized in that those components are withdrawn which will formspectroscopically detectable complexes with at least one reagent. 16.The method according to at least one of claims 1 to 15, characterized inthat components are withdrawn which have been unknown with respect totheir molecular nature, such as molecules, cells, vesicles, molecularcomplexes, which can be identified through an interaction with knownstructures, or through activities, such as enzymatic activity, orcomplex formation.
 17. The method according to claim 16, characterizedin that the unknown particles are pathogens or immunogens which areselectively withdrawn by collecting serums from at least one organismwherein at least one serum (serum 1) is obtained from the phase of anacute infection by a pathogen or immunogen which is optionally as yetunidentified (unknown), and at least one serum (serum 2) is obtainedfrom the same or at least one other organism with the same or ahomologous infection in the phase of chronical disease. wherein saidoptionally unknown pathogen or immunogen from serum 1 is induced tomeasurable complex formation with indirectly or immediatelyfluorescence-dye labeled antibodies from serum
 2. 18. The methodaccording to at least one of claims 16 to 17, characterized in that thesimultaneous binding of ligands giving different fluorescence signals,e.g. labeled antibodies from different organisms, is determined bycross-correlation.
 19. The method according to at least one of claims 16to 18, characterized in that the labeling of said antibodies is doneimmediately by at least one reaction with dyes capable of coupling, orindirectly by reaction with dye-labeled antibody binding domains, inparticular protein A derivatives or protein G derivatives.
 20. Themethod according to at least one of claims 1 to 19, characterized inthat said optionally unknown particles are per se known microorganismsor vesicles wherein the detected characteristics are specificinteractions with surface-expressed or cytosolic-expressed structuralelements of natural or recombinant proteins or peptides or enzymaticactivities with fluorescence-labeled target molecules.
 21. The methodaccording to at least one of claims 1 to 20, characterized in that saidmeasuring volume is composed of measuring subvolumes illuminated inparallel wherein the simultaneous illumination of a multitude ofmeasuring volumes is effected by at least one electromagnetic radiationsource using at least one holographic grid.
 22. The method according toclaim 21, characterized in that the registration of fluorescence signalsfrom at least one measuring volume is performed by confocal focussingusing a multitude of confocal pinhole apertures in the object plane, orby coupling the signals into optical waveguides in the object plane, orby multiarray detectors in the object plane.
 23. The method according toat least one of claims 21 to 22, characterized in that for performingparallelized measurements on at least two measuring volumes, at leasttwo measuring volumes in common or assembled in groups are focussedconfocally onto at least one detector element of a photon-registratingmeasuring element in the object plane in the signal registration. 24.The method according to at least one of claims 1 to 23, characterized inthat for detecting very low concentrations of fluorescing molecules, thesample volume is subjected to a scanning process prior to the actualmeasuring and withdrawal of at least one component wherein the timerequired for detecting a sought particle is shortened by varying thespace coordinates of the measuring volume with respect to the spacecoordinates of the sample volume continuously or discontinuously intime.
 25. The method according to at least one of claims 1 to 24,characterized in that the time interval δt for the measurement of one ormore volume elements having defined space coordinates prior to thedetection of a sought molecule by its fluorescence measuring signal isshorter than the average dwelling time of the sought molecule within ameasuring volume.
 26. A device comprising a receptor means having a poreand a measuring device for the illumination and/or measurement of ameasuring volume by electromagnetic radiation for performing the methodaccording to at least one of claims 2 to 25, characterized in that saidpore of the receptor means can be approached closely to the measuringvolume which is smaller than 10⁻¹⁴ l and said receptor means isconnected with a mechanically, optically or electrically controllablewithdrawal device.
 27. The device according to claim 26, consisting ofan arrangement of a closed or open container for receiving a samplevolume and at least one connection to at least one second volume elementwhich is in direct contact with the sample volume through an apertureand a liquid phase wherein said aperture is preferably immediatelyadjacent to said measuring volume.
 28. Use of the method according to atleast one of claims 1 to 27 for the preparative recovery of unidentifiedpathogens, immunogens or organisms which functionally express parts of agenome.
 29. Use of the method according to at least one of claims 1 to25 for the preparation of genetic probes for theidentification/detection/cloning of functional elements of a wholegenome and/or diagnostic and/or therapeutic agents derived therefrom.30. Use of the method according to at least one of claims 1 to 25 forthe analysis and preparative recovery of nucleated fetal cells frommaternal blood.
 31. Use of the method according to at least one ofclaims 1 to 25 for the detection and preparative recovery of at leastone specific gene of a microorganism the corresponding at least one geneproduct of which is presented on the inner or outer cell membrane orviral envelope.
 32. Use of the method according to at least one ofclaims 1 to 25 for the determination of the function of gene products ofdefined gene segments.